Development of new antitubercular agents is of critical importance worldwide. Our program has identified a new class of inhibitor of Mycobacterium tuberculosis(Mtb) that inhibits a novel protein not presently targeted by current antitubercular agents. The 2-alkoxy-carbonylamino-pyridines (2-ACPs) potently inhibit the growth of Mtb with an MIC99 (SRI-3072) as low as 0.15 microgram/ml (0.28 micromolar). Furthermore, SRI-3072 shows bactericidal activity, and shows significant activity in a murine-derived macrophage model with an EC90 & EC99 of 0.12 and 1.42 microgram/ml respectively. These analogs also show selective activity against Mtb versus a mammalian cell line. This program has successfully identified the target of these agents, the mycobacterial tubulin homolog FtsZ. The target protein has been cloned, expressed and isolated in quantities sufficient for development of in vitro polymerization and GTP hydrolysis assays. Three compounds, SRI-3072, SRI-76 14, and colchicine have been shown to inhibit polymerization of Mtb FtsZ in a dose dependent manner with IC50S of 50 uM, 60 uM, and 100 uM respectively. Furthermore. we have shown that SRI-7614 affects Mtb FtsZ polymerization by electron microscopy. SRI-7614 has also been shown to be active vs. a panel of single drug-resistant Mtb strains. We currently have crystal structures of Mtb FtsZ bound to citrate, GTPgS, and GDP. To date, about 200 2-ACP analogs have been screened in vitro against Mtb H37Rv. We have developed a SAR profile that will allow the preparation of more selective and more potent antitubercular agents. In this application, we propose to continue development of the 2-ACP class through preparations of new analogs of the more potent and selective subclasses, the 3-deaza-pteridines (priority), and the pyridodiazepines (backup). We will carefully evaluate these compounds for activity and selectivity in various in vitro assays including an in vitro Mtb H37Ra assay, an in vitro Mtb FtsZ polymerization and GTPase assay, an in vitro tubulin polymerization assay and a mammalian cell toxicity assay. Selected active agents will be further screened in an h in vitro macrophage model and a Mtb mouse model. The effect of inhibitors on FtsZ polymerization will be analyzed using electron microscopy. Data from the biological screening and the EM structural studies will feed back into compound design in an interactive, iterative drug design cycle that critically focuses on antibacterial potency and selectivity.